Among the most wide-spread and successful methods for the detection and analysis of RNA molecules is the so-called RT/PCR (reverse transcription/polymerase chain reaction). In this method, RNA molecules are transcribed into cDNA molecules by a reverse transcriptase (RT) and are then detected and/or analyzed by PCR-based methods.
Early researchers in this field have used either avian myeloblastosis virus RT or Moloney murine leukemia virus RT for reverse transcription. However, a significant problem in using RNA as a template for cDNA synthesis was the inability of these mesophilic viral RTs to synthesize cDNA through stable RNA secondary structures. To circumvent this problem, reverse transcription can be performed at increased reaction temperatures which can resolve secondary RNA structures, and additionally increase the specificity of primer extension. This requires the use of thermostable enzymes for reverse transcription, like Thermus thermophilus DNA polymerase (Tth pol) which is active at a temperature optimum of 70° C.
However, the use of thermostable enzymes for reverse transcription also generates new problems. In particular, when trying to detect and/or analyze low abundance RNA species in a biological sample, the thermostable enzyme for reverse transcription can catalyze the generation of non-specific reaction products during subsequent RT/PCR steps. This is especially the case in samples that have a large background of total RNA, and results in a substantial increase of the limit of detection of the target RNA to levels far above what can be necessary, for example in the quality control of biopharmaceutical products. To circumvent this problem, all handling and RT/PCR steps subsequent to reverse transcription have to be performed at elevated temperatures and under hotstart conditions. This, however, is often impractical and disadvantageous, especially when working in industrial scales.
An application wherein sensitive detection of low abundance RNA species, often in a large background of total RNA, is of particular relevance is the detection of a mycoplasma contamination in a biological sample.
Mycoplasmas are bacterial microorganisms with one of the smallest known genomes able to self-replicate. While most mycoplasmas are naturally harmless commensals, some of them are able to infect their natural hosts and cause diseases of varying severity. Mycoplasmas, particularly species of the genera Mycoplasma and Acholeplasma, are also frequent causes of contamination of primary and continuous cell lines and represent a serious problem for research and industrial laboratories involved in the development and production of cell-derived biological and pharmaceutical products. Thus, in spite of all preventive measures usually employed during the process of cell line propagation and handling, mycoplasma contamination continues to be a frequently occurring problem.
The analytical protocols recommended for mycoplasma testing of cell lines and cell-derived biological products include the use of broth/agar culture and indicator cell line tests. The broth/agar culture method is aimed at the detection of cultivable mycoplasmal agents, while the indicator cell line is used to detect fastidious non-cultivable mycoplasma species. Although the combination of these two methods enables efficient mycoplasma detection, the overall testing procedures are expensive, laborious and time-consuming (a minimum of 28 days). Moreover, there exists a number of non-cultivable mycoplasma species that are not amenable to this approach.
Novel mycoplasma testing methods based on the amplification of nucleic acids, in particular of mycoplasma derived ribosomal RNA have recently been developed. These methods have certain advantages with respect to costs, analytical sensitivity, simplicity and time. However, the detection limit of currently available methods is still above what is necessary in the quality control of biopharmaceutical products, especially when working with samples that have a large background of total RNA or other macromolecules.
Therefore, a need exists in the field to improve current RT/PCR methods to allow for improved detection limits of low abundance RNA species in biological samples.
The solution to the above problem is achieved by the embodiments characterized in the claims.